KR20230010715A - Systems and cartridges for sample testing - Google Patents

Abstract

A sample analysis system is provided to aid in diagnosis, and/or detection of the presence or absence, and/or identification of an analyte in a sample. A cartridge having reagents for isolating nucleic acids from a sample inserted into the cartridge and amplifying the isolated nucleic acids, and an instrument interacting with the cartridge are together a response system for detecting, identifying, distinguishing and/or quantifying target nucleic acids in a sample. provides a self-contained sample.

Description

Systems and cartridges for sample testing

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims priority to and benefit from U.S. Provisional Application No. 63/024,406, filed on May 13, 2020, the entire contents of which are incorporated herein by reference.

The subject matter described herein relates to a system for analyzing a sample, for example, to detect the presence or absence of a target analyte in the sample, and/or to determine the identity of the analyte in the sample. The system aids in the determination or diagnosis of a condition, disease, or disorder due to the presence of an analyte, eg, an infectious agent in a sample. The system includes a cartridge and an instrument that receives the cartridge and functions together to provide analysis of a sample inserted into the cartridge to detect, identify, differentiate and/or quantify the presence of a target nucleic acid in the sample.

In the analysis of a sample, determining the presence of a particular nucleic acid is desirable for a high level of accuracy and sensitivity. The availability of amplification techniques, such as polymerase chain reaction (PCR) and other nucleic acid amplification techniques, have made nucleic acid detection and discrimination a sensitive technique for analyzing pathogens or other agents in samples of biological origin. However, the need to perform multiple reagent steps, handle samples, and perform assays in a manner that avoids contamination, to achieve analytical accuracy, hampers the widespread application of these techniques outside sophisticated microbiology laboratories or clinical settings. interfere with

In a first aspect, a system comprising a cartridge and an instrument is provided. The cartridge comprises: (i) a plurality of chambers, the plurality of chambers including an extraction chamber and a detection chamber; (ii) a plurality of reagent canisters, each reagent canister of the plurality containing a reagent; and (iii) a plurality of magnetic particles once introduced into the extraction chamber and retained therein. The mechanism is configured to receive the cartridge and includes: (i) a first sonicator movable in at least one of the x-y-z coordinates; (ii) a magnetic field positionable to capture the plurality of magnetic particles in the extraction chamber; and (iii) an optical unit for illumination of the detection chamber and detection of signals therefrom.

In one embodiment, the cartridge comprises: (i) a plurality of chambers, the plurality of chambers including an extraction chamber and at least one detection chamber; (ii) a port for engaging a gas supply source; (iii) a plurality of reagent canisters, each reagent canister of the plurality containing a reagent; and (iv) a plurality of magnetic particles. In some embodiments, all or part of the reagent canister may be in fluid communication with a dedicated gas source through one or more gas supply ports. In one embodiment, the piercing element may be in fluid communication with a dedicated gas source through one or more gas supply ports. In some embodiments, the magnetic particles are in a first position on the cartridge and are movable into the extraction chamber. In another embodiment, the magnetic particles are in the extraction chamber prior to use of the cartridge and remain in the extraction chamber after use when the cartridge is deployed. In another embodiment, the magnetic particle is in a first position on the cartridge and is movable to the extraction chamber with the transfer of the fluid containing the sample inserted into the sample port on the cartridge when the fluid containing the sample is moved to the extraction chamber.

In one embodiment, each reagent canister includes a frangible material and includes or is configured to contact a piercing element. The piercing element, in one embodiment, is a component of a reagent canister. In another embodiment, the piercing element is a component of a cartridge or instrument and is positioned to contact the frangible material on the reagent canister. In one embodiment, the piercing element includes an opening in fluid communication with the reagent canister and/or a conduit connecting the reagent canister to the cartridge. In some embodiments, the opening of the piercing element forms a conduit having an inlet and an outlet. In use, gas enters the inlet of the piercing element through the gas supply port on the cartridge to displace reagents in the reagent canister for delivery to the cartridge through the outlet.

In one embodiment, the cartridge includes a port or a plurality of ports configured to engage a gas supply source. In another embodiment, the gas supply source is a pressurized gas source contained within a mechanism that receives the cartridge. In another embodiment, the gas supply source is a pressurized gas source external to instruments and systems.

In another aspect, a method for identifying (ie, detecting, discriminating, and/or distinguishing) the presence or absence of a target nucleic acid in a sample is provided. The method includes (i) providing an extraction chamber, including a detection chamber disposed downstream from the extraction chamber; (ii) moving the sample suspected of containing the target nucleic acid sample, the plurality of magnetic particles, and the fluid to the extraction chamber; (iii) entrapping, with a magnetic field, a plurality of magnetic particles complexed with nucleic acids in an essentially or substantially fluid-free extraction chamber and introducing a gas at a temperature of at least about 35° C. into the extraction chamber; (iv) introducing a volume of elution medium into the extraction chamber; (v) releasing a plurality of magnetic particles complexed with the nucleic acid from the magnetic field into the elution medium to release the nucleic acid from the plurality of magnetic particles; (vi) trapping the plurality of magnetic particles with a magnetic field to maintain the plurality of magnetic particles in the extraction chamber; (vii) moving the elution medium and the nucleic acid from the extraction chamber to a downstream chamber for contact with reagents for amplification of the nucleic acid; and (viii) amplifying the nucleic acid and detecting the amplification product in the detection chamber.

In another aspect, a kit comprising a cartridge and a pipette is provided. In some embodiments, the pipette includes an overflow chamber. In another embodiment, the pipette is configured to dispense a specific fixed and known volume of sample, such as a patient sample, into the cartridge. In one embodiment, the cartridge contains reagents for isolating target nucleic acids from a sample and amplifying the target nucleic acids, if present. The cartridge is insertable into an instrument configured to detect the presence (or absence) of amplicons of a target nucleic acid and report the results to a user of the cartridge.

1 shows a testing architecture or system comprised of an instrument, an optical source, and a cartridge, in accordance with some embodiments.
2A-2D show different views of a cartridge for sample testing, according to some embodiments.
3 shows a chamber and mechanism for unloading a canister containing reagents into a cartridge for sample testing, according to some embodiments.
4 shows a valve used to control fluid flow in a cartridge for sample testing, with dimensions shown in units of mm, according to some embodiments.
5 shows a partial view of a detection chamber panel of a cartridge for sample testing, in accordance with some embodiments.
6A-6D show components of an optical coupler for use in an optical unit for an instrument, according to some embodiments.
7A-7AF show a series of steps in a sample testing method using a cartridge, in accordance with some embodiments.
8 is a flow diagram illustrating steps of a sample testing method, in accordance with some embodiments.
In the drawings, elements having the same or similar numerals may refer to elements having the same or similar characteristics, unless otherwise stated.

In the detailed description that follows, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be apparent to those skilled in the art that embodiments of the invention may be practiced without these specific details. In other instances, well known structures and techniques have not been shown in detail in order not to obscure the present invention.

In the field of nucleic acid detection, the ability to amplify a target sample at an exponential rate using techniques such as polymerase chain reaction (PCR), or isothermal processes such as helicase dependent amplification, has significantly increased the sensitivity of detection. However, the delicate balance of several reagent steps free from contamination has hindered the application of these techniques to compact, easy-to-use instruments that produce accurate results in limited time.

The systems, assays, devices, methods, and kits described herein provide for the qualitative detection and/or discrimination of a variety of different analytes, e.g., pathogens, target nucleotides that may be associated with pathogens, bacteria, viruses, fungi, or other microorganisms. can be used for For example, in some embodiments, the technology relates to rapid multiplex real-time PCR (RT-PCR) assays for the qualitative detection and discrimination of nucleotides from pathogens of interest. In some embodiments, pathogens of interest may include pathogens, bacteria, viruses, fungi, or other microorganisms and infectious agents. In one embodiment, the pathogen of interest is a virus such as influenza A (Flu A), influenza B (Flu B), respiratory syncytial virus (RSV) or SARS-CoV-2.

The systems, assays, devices, methods and kits described herein provide for the analysis of a variety of nucleic acids of interest, such as DNA and/or RNA. In some embodiments, the nucleic acid of interest is viral RNA extracted from nasal and nasopharyngeal swabs in a viral transfer medium. In some embodiments, the analyzed specimen, including nasal and nasopharyngeal swabs, is from a patient with signs and symptoms of a respiratory viral infection.

The systems, assays, devices, methods and kits described herein also, in some embodiments, provide the user the flexibility to select which results can be reported, such as identification of the presence or absence of viral RNA. Thus, in some embodiments, the systems, assays, devices, methods and kits described herein provide in vitro diagnostic tests intended to aid in the differential diagnosis of diseases, such as viral diseases. In some embodiments, the viral disease of interest includes, but is not limited to, Flu A, Flu B, RSV and SARS-CoV-2. The techniques presented here can provide information related to infections, such as viral infections in humans, along with clinical and epidemiological risk factors.

In some embodiments, the techniques provided herein are compliant with the Clinical Laboratory Improvement Amendment (CLIA) of 1988, 42 U.S.C. Includes testing performed by laboratory personnel, such as laboratory personnel certified under §263a. In other embodiments, the systems, assays, devices, methods and kits described herein may be distributed and used in other settings, such as patient care settings outside of a clinical laboratory environment.

In some embodiments, the systems, assays, devices, methods, and kits described herein provide results indicating positive or negative results for the detection of a pathogen of interest, such as bacteria, viruses, fungi, or other pathogens or microorganisms. In some embodiments, a positive or negative result may indicate the presence of a viral infection, such as the presence of Flu A, Flu B, RSV or SARS-CoV-2. In some embodiments, positive or negative results provided by the techniques provided herein may be considered along with clinical correlation of patient history and other diagnostic information that may be needed to determine patient infection status. For example, a positive or negative result for one pathogen, such as a viral pathogen, does not exclude the possibility of further infection, such as bacterial infection or co-infection with another virus.

In some embodiments, the techniques provided herein may provide information related to infection by a novel pathogen, such as, for example, a novel influenza virus. In such cases, specimens should be collected and processed according to appropriate safety, documentation and submission guidelines.

In some embodiments, the systems, assays, devices, methods, and kits described herein provide for determining the presence or absence of influenza virus nucleic acids in a sample, such as a nasal or nasopharyngeal swab, from a patient suspected of having a pathogenic infection and/or disease. . Influenza virus is the causative agent of highly contagious acute viral infections of the respiratory tract. Influenza viruses are immunologically diverse, monotonic RNA viruses. There are three types of influenza viruses: A, B and C. Type A viruses are the most widespread and are associated with the most serious infectious diseases. Type B viruses generally cause milder illness than type A viruses. Type C viruses have not been associated with large-scale epidemics of human disease. Both type A and type B can be prevalent simultaneously, but usually one type dominates during a given season. Each year in the United States, an average of 5% to 20% of the population gets influenza; More than 200,000 people are hospitalized for complications of influenza; And, about 36,000 people die from influenza-related causes. Some people, such as adults over the age of 65, children, and people with certain health problems, are at high risk for serious influenza complications.

In some embodiments, the systems, assays, devices, methods and kits described herein can be used to detect the presence or absence of SARS-CoV-2 viral nucleic acid in a sample such as a nasal or nasopharyngeal swab from a patient suspected of having a pathogenic infection and/or disease. provide a decision SARS-CoV-2, also known as the COVID-19 virus, was first discovered in December 2019 in Wuhan, Hubei Province, China. The virus is thought to have originated in bats, like the novel coronaviruses SARS-1 and MERS, but SARS-CoV-2 may have had intermediate hosts such as pangolins, pigs or civets. The WHO declared COVID-19 a pandemic on March 11, 2020, and human infection has spread globally, with hundreds of thousands of confirmed infections and deaths. The median incubation period is estimated at 5.1 days, and symptoms are expected to appear within 12 days of infection. Symptoms of COVID-19 are similar to other viral respiratory illnesses and include fever, cough and shortness of breath.

In some embodiments, the systems, assays, devices, methods and kits described herein may be used for the presence or absence of human respiratory syncytial virus (RSV) nucleic acid in a sample such as a nasal or nasopharyngeal swab from a patient suspected of having a pathogenic infection and/or disease. Provides a determination of absence. RSV is a negative monotonic RNA virus of the Paramyxoviridae family. RSV is a leading cause of lower respiratory infections and hospital visits in infancy and childhood. In the United States, 60% of infants are infected in their first season of RSV and almost all children become infected with the virus by the age of 2-3 years. 2-3% of those infected with RSV develop bronchiolitis and require hospitalization. Natural infection with RSV induces protective immunity that weakens over time—perhaps more than other respiratory viral infections—so people can become infected multiple times. Occasionally, infants may become symptomatic more than once within a single RSV season. Severe RSV infections are increasingly found among elderly patients.

In some embodiments, the systems, assays, devices, methods, and kits described herein employ a nucleic acid amplification process, such as real-time PCR or other amplification techniques, used in conjunction with an instrument to detect and differentiate target nucleotides in a sample inserted into a cartridge. It provides an approach to a single, disposable, stand-alone assay cartridge with reagents for In some embodiments, the target nucleotides detectable by the systems, cartridges and methods provided herein are nucleotides of pathogens and/or pathogens, such as bacteria, viruses or fungi. For example, in some embodiments, the nucleotides analyzed, detected and/or distinguished may include DNA and/or RNA, such as RNA from influenza A, influenza B, RSV, and/or SARS-CoV-2. there is. Other exemplary target nucleic acid analytes include Bordetella pertussis, Bordetella parapertussis, C. difficile, Group A β-hemolytic streptococcus ( β-hemolytic Streptococcus (Streptococcus pyogene), pyogenic group C/G (Streptococcus dysgalactiae), herpes simplex virus 1, herpes simplex virus 2, varicella-zoster virus, human metapneumovirus, trichomonas, human adenovirus and parainfluenza virus (PIV-1, PIV-2, and/or PIV-3 ), including those from A stand-alone cartridge may contain reagents for detection and/or discrimination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more target analytes.

In some embodiments, results indicating the presence or absence of multiple target nucleotides may be provided from analysis of a single patient sample, such as a nasal or nasopharyngeal swab. For example, in some embodiments, a single sample can be tested for the presence or absence of multiple pathogen targets in a single sample, such as multiple bacterial, viral, and/or fungal nucleotides, and/or mixtures and combinations thereof. It can be assayed in disposable, and stand-alone cartridges. In some embodiments, a single sample can be assayed with a single cartridge, as described herein, for the presence of multiple viral nucleotides, the viral target comprising influenza A, influenza B, RSV and/or SARS-CoV-2. It contains four different viral RNA targets that

In some embodiments, the systems, assays, devices, methods, and kits described herein extract, amplify, or extract viral RNA or DNA present from a sample, such as a nasal cavity, nasopharynx, sputum, or blood sample, obtained from a symptomatic patient. and detection. In some embodiments, the techniques provided herein can perform a complete assay, including extraction, amplification, and detection, in less than about 1 hour, less than about 30 minutes, and/or less than about 20 minutes, such as about 22 minutes.

In some embodiments, sample analysis by the systems, assays, devices, methods, and kits described herein may be performed on a transfer medium, such as a viral transfer medium, such as a sample collected on a nasal passage or swab placed in the mouth or throat. It begins by placing a patient sample. In some embodiments, the transport medium containing the sample sample extracted from the swab is transported to the liquid sample add port, or sample port, of the cartridge described herein. In another embodiment, the swab is inserted directly into the cartridge to process the sample on the swab, as described herein.

In some embodiments, the transfer medium is delivered to the sample port of the cartridge with a transfer pipette supplied as part of a kit with the cartridge. In some embodiments, a provided transfer pipette comprises an overflow chamber and is configured to transfer and/or dispense a specific, fixed and known volume of sample, such as a patient sample extracted from a nasal cavity or nasopharyngeal swab specimen, to a transfer medium. . In some embodiments, a provided transfer pipette is a sample between about, 50-2000 μL, 50-1000 μL, 100-500 μL, 150-400 μL, 175-350 μL, 200-300 μL, 225-275 μL, or configured to transfer a volume of sample of about 150 μL, about 200 μL, about 250 μL, about 300 μL or about 350 μL.

In some embodiments, after the sample is introduced into the sample port of the cartridge, the port is closed and the cartridge is inserted into the instrument to initiate sample processing. In some embodiments, as detailed herein, the sample is pushed out of the sample port by the lysis buffer. In some embodiments, the lysis buffer also rehydrates a process control, such as an Escherichia virus MS2 (MS2) process control. In some embodiments, the sample and process controls are moved along with particles or beads, such as magnetic particles, into an extraction chamber of a cartridge as described herein. In some embodiments, a solution containing the sample, optional process controls, and fluid (such as a lysis buffer or transport medium) is mixed in an extraction chamber, and cells or organisms in the sample are further lysed by the mixing. In one embodiment, mixing is by sonication of the extraction chamber. In some embodiments, beads having sample DNA and/or RNA associated with them are washed, and DNA and/or RNA is eluted from the beads. In some embodiments, a solution containing purified and/or isolated DNA and/or RNA is used to rehydrate a lyophilized master mix containing reagents for amplification of DNA and/or RNA. In one embodiment, a solution containing the isolated and/or purified DNA and/or RNA is passed from the extraction chamber to a plurality of reagent chambers, each reagent chamber in dedicated fluid communication with the detection chamber. Each reagent chamber contains reagents for amplification and detection of a specific DNA or RNA target analyte. In this way, cartridges with 2, 4, 6, 8, 10, 12 or any number of reagent chambers with dedicated detection chambers achieve multiplexing of target nucleic acids from a single sample. In one embodiment, the cartridge includes four reagent chambers, each with a dedicated detection chamber, each reagent chamber containing reagents (e.g., primers, probes, enzymes, salts, sugars) for amplification and detection of a particular target analyte. , etc.). In one embodiment, each reagent chamber contains reagents (also referred to in the art as a master mix) for amplification and detection of one of the nucleic acids from a specific pathogen. In one embodiment, the pathogen is selected from influenza A, influenza B, RSV, and SARS-CoV-2. In one embodiment, the pathogen is influenza A, influenza B, RSV, and SARS-CoV-2, Bordetella pertussis, Bordetella parapertussis, C. difficile, Group A β-hemolytic streptococcus ( Streptococcus piogen), pyogenic group C/G (Streptococcus disgalactie), herpes simplex virus 1, herpes simplex virus 2, varicella-zoster virus, human metapneumovirus, trichomonas, human adenovirus and Parainfluenza viruses (PIV-1, PIV-2, and/or PIV-3). In some embodiments, reagents in each reagent chamber are transferred to dedicated detection chambers in which amplification of target nucleic acid sequences can be performed. In some embodiments, amplification in a detection chamber is a Taq-man® multiplex multiplex real-time RT-PCR reaction performed under optimized conditions to generate amplicons for the target virus (if present) and process controls present in the sample. can include In one embodiment, each detection chamber is provided with an optical window for interrogation by an optical system of an instrument housing a cartridge to inspect amplicons in the detection chamber to determine the presence or absence of a particular label, tag, or detection reagent. It consists of

In some embodiments, each master mix contains primers and labeled probes, such as dual-labeled, that are unique to one, two, or more viral targets and/or process controls. In some embodiments, the probe is labeled, for example, with a fluorophore on one end and a quencher on the other end. In some embodiments, master mix reagents include cDNA of viral RNA, if present, and reagents for a reverse transcriptase step to optimally generate MS2 bacteriophage process control RNA. In some embodiments, a polymerase separates a fluorophore from a quencher by cleaving a probe bound to a complementary DNA sequence during DNA amplification. In some embodiments, this cleavage produces an increase in fluorescence signal and when sufficient fluorescence is achieved, the sample is reported as positive for the detected target sequence. In some embodiments, the device receiving the cartridge also includes a user interface screen for controlling, monitoring, and reading results such as positive, negative, and/or invalid results for the presence or absence of a target nucleotide sequence.

1 shows a testing architecture 102 that includes an optical source 122 and a mechanism 104 for receiving a cartridge 112 in accordance with some embodiments. Instrument 104 is configured to receive a cartridge 112, which cartridge contains a sample suspected of containing a target nucleic acid. In some embodiments, a target nucleic acid is part of a nucleic acid of a pathogen or is associated with a pathogen product or protein. Pathogens may include bacteria, viruses, prions, spores, and the like.

With continuing reference to FIG. 1 , instrument 104 further includes one or more sonicators, shown in FIG. 1 as sonicator 110 , and movable in at least one of the x-y-z coordinates. One or more sonicators may be selected portions or portions of the cartridge 112 (e.g., to enhance interactions of the components of the chamber and/or to provide mixing for chemical interactions or reactions therebetween). eg, an extraction chamber, a detection chamber, a sample chamber, a combination of chambers, or the like). In one embodiment, the cartridge includes a wall made of a flexible material, and the cartridge is positioned such that one or more sonicators can contact the flexible wall when inserted into an instrument. In one embodiment, the cartridge is constructed of a rigid plastic or thermoplastic material, and the flexible material is attached to one side of the rigid cartridge, thereby creating an extraction chamber with a rigid wall on one side of the cartridge and a flexible wall on the other side of the cartridge. form A sonicator of the instrument, movable in at least one of the x-y-z coordinates, is movably positioned to sonicate at one or more locations on the flexible wall of the extraction chamber.

In some embodiments, instrument 104 also includes at least one magnetic field 116, such as an electromagnet, solenoid, or any element that generates a movable and/or switchable (on/off) local magnetic field. In one embodiment, the local magnetic field may be positioned to trap a plurality of magnetic beads or particles in a cartridge, such as an extraction chamber of the cartridge. The magnetic particle, as attached to its surface, may contain portions of nucleic acids extracted from cellular or pathogenic components of the sample.

In some embodiments, the instrument includes a pneumatic unit 114 that provides pressurized fluid to move liquids, such as reagents and eluents, into the cartridge and from one location on the cartridge to another. The pneumatic unit 114 may also use pressurization/decompression to remove reaction residues from the cartridge. For this purpose, the pneumatic unit 114 may use a pump, a pressurized gas cartridge, or the like, and may be coupled with a pressurized gas line or gas line of the facility. The pneumatic unit 114 may be coupled to the cartridge 112 via a fluid (or gas) manifold (not shown) coupling the fluid source of the pneumatic unit to a valve or conduit of the cartridge. In some embodiments, instrument 104 activates piercing elements (eg, to open a reagent canister in a cartridge), or cartridges, such as pins and other elements, to open/close valves of chambers and fluid conduits of the cartridge. and an actuator 118 that mechanically activates the components of 112.

With further reference to FIG. 1 , in some embodiments, testing architecture 102 may include an optical unit 126 for illumination of a detection chamber of a cartridge and detection of a signal therefrom. In some embodiments, the optical unit is part of instrument 104 . The optical unit may include an optical source 122 that provides an excitation signal 124 that is passed through an optical coupler 128 to the detection chamber of the cartridge 112 . The optical coupler 128 may also include one or more detectors that provide a response signal 120 from the detection chamber back to the processor circuit 106 of the instrument. Response signal 120 may be an electrical signal converted from a photosensitive element of optical coupler 128, which indicates the presence of the suspected target nucleic acid in the sample. In some embodiments, excitation signal 124 is selected to derive a desired response signal 120 from a suspect target nucleic acid. For example, in some embodiments, excitation signal 124 is an optical pump for fluorescence emission of a tag that has chemical affinity with at least a portion of a suspected target nucleic acid. Thus, a response signal 124 may be provided when fluorescence emission is detected in the appropriate spectral band of the tag.

Referring further to FIG. 1 , processor circuit 106 executes instructions stored in memory circuit 108 in instrument 104 . As a result, instrument 104 may at least partially perform one or more steps of a method as described herein. For example, the processor circuit 106 may perform steps for signal processing of the response signal 120 . Processor circuit 106 may also control pneumatic unit 114, sonicator(s) 110, and optical unit 126 to perform steps as described in the methods described herein.

2A-2D show different views of a cartridge for sample testing in accordance with some embodiments. 2A shows a top view of a cartridge 2100 for sample testing, in accordance with some embodiments. The cartridge includes a sample port 2102, an extraction chamber 2104 coupled with an extraction chamber outlet valve 2105, and one or more PCR/detection chambers 2106, 2108, 2110, and 2112 (i.e., in some embodiments the chambers are PCR may be configured to serve as both a chamber and a detection chamber), and one or more PCR/detection chamber valves 2114, 2116, 2118, and 2120. In some embodiments, the orientation of the cartridge when inserted into the instrument with the sample port positioned below the centerline provides for movement of the sample from the sample port into the extraction chamber in the opposite direction of gravity. In some embodiments, a cartridge may include one or more reagent canisters 2122, 2124, 2126, and 2128, at least some of which may include an extraction chamber, and one or more reagent canister valves 2130, 2132, 2134, and 2136. ) can be in fluid communication with. In some embodiments, sample port 2102 receives a swab containing a sample (eg, a biological sample). In some embodiments, the second sample port may be configured to receive a liquid sample (eg, from a drop, or syringe, and the like). In some embodiments, a biological sample may include bodily fluid (eg, blood, serum, plasma, sputum, mucus, saliva, tears, feces, or urine). In some embodiments, the biological sample is human, and the presence of one or more portions of the target nucleic acid may represent a medical diagnosis for the individual providing the sample. Each reagent canister 2122, 2124, 2126 and 2128 contains reagents that may be lyophilized or may be in liquid form. In some embodiments, reagents in at least one reagent canister may include a dissolution medium in fluid communication with the sample ports through canister valves 2130, 2132, 2134, and 2136. Once in solution, the reagent reacts with the sample components and other reagents to extract a portion of the target nucleic acid that may be present in the sample. The volume of each reagent canister may be equal to, or approximately equal to, the volume of the extraction chamber.

In some embodiments, a cartridge includes several pieces assembled together. The first piece may comprise an injection molded plastic piece containing several chambers and conduits. Other pieces may include a flat cover, or plastic film, with functions for aligning the instrument's actuators, and sample port cover attachment points (see eyelets and other features). In some embodiments, one or more fluid conduits and chambers of the cartridge may include a hydrophobic filter to facilitate metering of fluid and to facilitate complete filling and evacuation of the detection chamber. In some embodiments, a hydrophobic filter may be welded to the cartridge body. In some embodiments, the cartridge may include one or more bubble traps 2166, 2168, 2170, and 2172 to trap air bubbles formed during the workflow (see 'shark fin' feature). In some embodiments, action of the sonicator in the device is against a plastic film.

In some embodiments, the extraction chamber includes a first inlet port at a first location and a second inlet port at a second, different location. In some embodiments, one of the first inlet port and the second inlet port may be located at or below a centerline separating the brewing chamber into two sections of essentially equal volume or two sections of unequal volume. there is. In some embodiments, when inserted into an instrument with a sample port positioned below the centerline, the orientation of the cartridge provides for movement of the sample from the sample port into the extraction chamber in the opposite direction of gravity.

With further reference to FIG. 2A , the cartridge has a plurality of supply ports such as ports 2138, 2140, 2142 and 2144, a gas vent 2146, a vent valve 2152, and after reagents have been used and removed from the fluid conduit of the cartridge. It may include a plurality of gas supply ports, such as a waste path 2148 fluidly coupled by a waste valve 2150 with a waste chamber 2164 to collect reagent residues. In some embodiments, all or a portion of the reagent canister may be in fluid communication with a dedicated gas source through one or more gas supply ports. In some embodiments, the instrument's pneumatic unit moves fluid, such as a sample or fluid containing fluid processing reagents, and the like, through the cartridge and from the extraction or processing chamber to the waste chamber via the waste pathway when the waste valve is open. A pressurized gas is supplied to provide reaction products or reaction residues. Gas supply ports 2138, 2140, 2142, 2144 couple the fluid conduit of the cartridge with the supply line of the pneumatic unit. In some embodiments, the pneumatic unit may provide a gas supply to the cartridge through a supply line of the pneumatic unit coupled to a gas supply port of the cartridge. In some embodiments, the pneumatic unit may provide a gas supply to the cartridge at about 2 - 70 kPa (kilo-Pascals), 2 - 50 kPa, 2 - 35 kPa, 5 - 50 kPa, 5 - 35 kPa or 2 - 150 kPa. In some embodiments, the fluid is a gas, and in some embodiments, the gas can be air, nitrogen, argon or any other inert gas or combination thereof. The system's gas vent and vent valve can act as a low pressure point to relieve gas pressure and direct gas flow from the gas supply port through one or more chambers of the cartridge.

With continued reference to FIG. 2A , in some embodiments, a cartridge includes a canister or chamber containing beads or particles, such as magnetic beads or paramagnetic particles (PMPs). In one embodiment, the magnetic particles are introduced into the extraction chamber and, once introduced into the extraction chamber, remain therein for the remainder of the process, as described in more detail below. Selected portions of target nucleic acids from cells or other pathogenic components of a sample may attach to the surface of a bead or particle, in concert with reagents from a reagent canister, if the target nucleic acid(s) are present, as further described below. Separation and/or purification from the sample is provided.

With further reference to FIG. 2A , in some embodiments, the extraction chamber functions as a metering chamber for receiving and/or dispensing an accurate measure of liquid. In this embodiment, a liquid, such as an elution medium, held in a reagent canister or other chamber of a cartridge is moved to the extraction chamber by a channel or conduit fluidly connecting them. The extraction chamber has a pre-selected volume formed, which, when exceeded, overflows into the overfill chamber 2174, thus ensuring the volume of fluid formed and metered in the extraction chamber. In one embodiment, the metering function of the extraction chamber is used when a fluid such as an elution medium is introduced into the extraction chamber. The elution medium is conveyed to the detection chamber on the cartridge downstream of the reagent chamber and extraction chamber, and the nucleic acid(s) in the elution medium are amplified for detection. In some embodiments, it is desirable to have a known volume of elution medium with the nucleic acid placed in each amplification or detection chamber to obtain quantitative results. In this embodiment, the elution medium is passed through conduits available by valves 2114, 2116, 2118, and 2120 to a detection or reaction chamber for nucleic acid amplification. In some embodiments, the volume of elution medium in the reagent canister may be greater than the preselected volume of the metering chamber. Thus, the amount of elution medium delivered to the extraction chamber is not less than the volume of the preselected metering chamber. In an embodiment, the volume of elution medium in the reagent canister is greater than the volume of the portion of the extraction chamber that serves as the metering chamber to allow elution medium to flow into the overfill chamber to ensure a known measured volume of elution medium. In some embodiments, overfill chamber 2174 includes a sponge-like material to capture the overflow of media. In some embodiments, filter paper or other cellulosic material may be used as an absorbent for excess effluent media. Thus, it is desirable to avoid dripping or flowing back from the overfill chamber 2175 into the fluid conduit of the cartridge. In another embodiment, the overfill chamber 2174 is in fluid communication with the extraction chamber and the liquid medium reagent canister to allow a known volume of reagent medium to flow into the extraction chamber. In some embodiments, overfill chamber 2174 is in fluid communication with extraction chamber 2104 and allows a known volume of liquid medium to remain in the extraction chamber. In some embodiments, the volume of liquid medium in at least one of reagent canisters 2122 , 2124 , 2126 , and 2128 exceeds the volume of extraction chamber 2104 . Excess liquid is removed from the brew chamber 2104 through the brew outlet valve 2105 and the overflow valve 2152.

With continued reference to FIG. 2A , cartridge 2100 is indicated by identifiers 2154, 2156, 2158 and 2160 and contains a dried or lyophilized master mix (MMX), referred to as a reagent chamber or PCR reagent chamber. It includes a chamber that Beads or lyophilates of amplification reagents (eg, primers, probes, enzymes, salts, sugars, etc.) for amplification of target nucleic acids are placed in the reagent chamber. In some embodiments, the lyophilizates in each reagent chamber may be different from each other. Without loss of generality, several combinations may be realized, where two or more reagent chambers may be dedicated to the same lyophilisate (e.g., a 2/2 combination) or three chambers may be dedicated to the same lyophilisate (e.g., a 3/2 combination). 1 combination), or each chamber can have a different lyophilizate. In some embodiments, the reagent chamber may have a bulbous shape, and the lyophilizate may be bead-shaped, or any other shape having a smaller diameter than the diameter of the bulb-shaped chamber (e.g., hemisphere, disk, ellipses, and the like). In one embodiment, the lyophilizate contains reagents for process control that act as internal controls for the amplification process. The process control reagent may be, for example, a reagent disposed in the chamber of the cartridge in dry or liquid form that is normally transferred to the extraction chamber as a liquid. In some examples, the process control reagent may be lyophilized Escherichia virus MS2 (MS2) bacteriophage process control. In some embodiments, a sonicator of the instrument is positioned in contact with the cartridge to facilitate mixing of the nucleic acid and reagents in the PCR reagent chamber. Each reagent chamber is in fluid communication with a detection chamber in which thermal cycling can occur and nucleic acids are amplified. Amplicons in each detection chamber have detectable labels for detection, identification and/or differentiation. The detectable label or tag may be a fluorescent emissive tag or other radioactive emissive tag attached to the amplicon. Without loss of generality, any number of detection chambers for nucleic acid amplification may be realized in various embodiments.

In some embodiments, a metered volume or amount of sample suspected of containing the target analyte is transferred from the sample chamber through a sample port along with a liquid such as a lysis buffer. The liquid may contain or be passed through a chamber with a drying process control to rehydrate process control, such as an MS2 bacteriophage process control. In some embodiments, the weighed sample along with process controls and magnetic beads are moved to the extraction chamber. In some embodiments, a solution comprising the sample, process controls and magnetic beads are mixed by sonication, and cells in the sample are lysed to release nucleic acids into the solution in the extraction chamber. In some embodiments, the lysis buffer is removed from the extraction chamber, and the magnetic beads and any nucleic acids attached to the magnetic beads are immobilized in the extraction chamber by a magnetic field applied to the walls of the chamber. One or more different wash solutions may then be sequentially introduced into the extraction chamber and the magnetic beads ejected into the wash solution. After the washing step is completed and the washing solution is drained from the extraction chamber, the elution buffer is introduced into the extraction chamber and the magnetic beads are fixed in the extraction chamber by a magnetic field applied to the chamber wall. The magnetic beads are released into the elution buffer to elute nucleic acids from the magnetic particles into the elution buffer. In some embodiments, an elution buffer comprising nucleic acids isolated from the sample, and optionally process control nucleic acids, is used to rehydrate the lyophilized master mix in each PCR reagent chamber.

In some embodiments, the cartridge may include liquid sample and/or swab sample presence detection function(s). Thus, a signal is passed to the processor circuitry of the device when the detection function indicates the presence of a sample in the cartridge and/or a swap in the cartridge. Upon receipt of a signal confirming the presence of a sample and/or swap of a cartridge, a program stored in the instrument may automatically become a sequence of events to determine the presence or absence of a target analyte in the sample. In some embodiments, the instrument may provide a warning notification to the user indicating that the cartridge is ready and that the testing sequence may begin.

The cartridge may contain a control sample in a separate chamber or an internal control placed in its own dedicated chamber that may or may not be sent to the extraction chamber for processing. Thus, a control sample can include a nucleic acid control that has undergone one or more or all of the processing steps of a regular sample, including attaching magnetic beads that undergo chemical interaction with at least one or all of the reagents comprising the elution medium. .

In some embodiments, the cartridge may include an insertion feature that facilitates a mechanism to pull the cartridge within the system, such as a tab, notch, divot, or similar form of physical fiducial. In some embodiments, when the cartridge is inserted into the instrument and during sample processing, the orientation of the cartridge is such that the sample port is below the center line intersecting the cartridge at the insertion and processing location. In this embodiment, the opposite direction of gravity transfers the sample from the sample port to the extraction chamber.

2B-2D illustrate a profile or configuration of a cartridge for sample testing according to one embodiment. FIG. 2B shows a plan view of a first side of the cartridge, which may be the 'inner' side of the cartridge relative to the instrument. 2C shows a plan view of a second side of the cartridge, the first side and the second side being opposite and complementary to each other. Extraction chamber 2104, reagent canisters 2122, 2124, 2126, and 2128, detection chambers 2106, 2108, 2110, and 2112, waste chamber 2164, bubble traps 2166, 2168, 2170, and 2172, and waste. Path 2148 is as described with respect to FIG. 2A. In the embodiment shown in FIGS. 2B-2D , the cartridge includes two sample ports, a first sample port 2102 designed to receive a swab with a sample thereon, and a second sample port 2200 It is configured to receive a liquid sample such as a pipette. A hydrophobic filter 2202 may be disposed in the conduit leading to the detection chamber (detection chamber panel 2204) to facilitate fluid flow and prevent residue from remaining in the conduit. Alignment feature 2206 allows positioning of the cartridge within the instrument.

As shown in FIG. 2C, the cartridge may include an overfill chamber 2174 (optional) and a chamber 2152 containing magnetic beads. Liquid port 2200 serves as a sample port for liquid samples (eg, the sample port of FIG. 2A ). In some embodiments, liquid port 2200 may include a frustrated total reflection feature 2300 that enables detection of the presence of a liquid sample (and thus activates a fluid conduit associated with liquid port 2200). there is. For example, in some embodiments port 2200 is designed such that when an optical beam passes or crosses port 2200, detection of liquid is achieved if a sample is in the beam path.

FIG. 2D shows a perspective view of the cartridge with the inside of FIG. 2B being foreground; 2D is a side view showing the plastic film 2400 covering the inside of the cartridge (see FIG. 2B) and the cartridge body 2042. In some embodiments, the cartridge body is about 20 mm wide (eg, 23.5 mm wide) and the plastic film is about 1 mm thick, as also shown in FIG. 2D .

3 illustrates a reagent canister 3100 (like the exemplary reagent canisters 2122, 2124, 2126, or 2128 from FIG. 2A or 2C) and a mechanism for dispensing reagents in the canister into cartridges, in accordance with some embodiments. show In some embodiments, reagents in all or part of the plurality of reagent canisters (eg, one of 2122, 2124, 2126, or 2128 from FIG. 2A or 2C) are liquid reagents. In some embodiments, reagent canister 3100 includes a frangible membrane 3102 and a piercing element 3104 positioned to pierce frangible membrane 3102 . An actuator 3108 of a mechanism that receives the cartridge may press against the canister 3100 to pierce the frangible membrane 3102 to dispense the reagent therein, causing it to move against the piercing element 3104. do. Actuator 3108 may include a solid pin pressed against the canister by an electric motor of the appliance. In some embodiments, the piercing element 3104 may be formed (eg, by injection molding) into the cartridge body; That is, the piercing element 3104 is positioned on the cartridge for contact and penetration with the frangible membrane 3102 on the reagent canister 3100. In some embodiments, the piercing element may be part of an actuator, part of an instrument, part of a cartridge, or part of a reagent canister. In some embodiments, the frangible membrane or frangible material is an aluminum laminate, diaphragm, valve, port, or similar (e.g., any element that can be pierced or pierced open without necessarily bursting or breaking). ) can be. In some embodiments, frangible membrane 3102 may be a laminate of multi-layer materials. A headspace may exist between the piercing element 3104 and the frangible membrane 3102, and deflection by the actuator 3108 moves the reagent canister into the headspace and brings it into contact with the piercing element. Alternatively, there may be a headspace between the reagent canister and the actuator, wherein the piercing element is in contact or near contact with the frangible material, and movement of the actuator across the headspace to contact the reagent canister causes the piercing element to break. Penetrates fragile materials. Once the integrity of the frangible material is destroyed, reagents in the reagent canister may be released.

In some embodiments, the piercing element 3104 may include a hollow conduit that fluidly couples the inlet 3110 with the outlet 3112 once the frangible membrane 3104 is pierced. In some embodiments, a valve 3114 at the inlet or outlet may further control the flow of reagent 1306 out of canister 3100 once actuator 3108 ruptures frangible membrane 3102. there is. In some embodiments, gas from the dedicated gas source is travelable from the gas source to the reagent canister 3100 through a hollow conduit. In some embodiments, each reagent canister interacts with one actuator of the instrument. In some embodiments, each reagent canister interacts with a dedicated actuator. In some embodiments, when the actuator is not activated and the frangible membrane is intact, valve 3114 can be opened, allowing gas flow to flow from the inlet to the outlet through the reagent chamber and the hollow piercing member. This may be useful to remove reagents and/or residual components of the extraction chamber from a previous step prior to activating the piercing mechanism to transfer the reagent solution to the extraction chamber.

4 shows a valve used to control fluid flow in a cartridge, in accordance with some embodiments. In some embodiments, the valve is a pinch valve that includes a film weld energy director 4100 , a valve seat 4102 , and a valve cavity 4104 . The cross-sectional view shows some exemplary dimensions (mm) of the valve seat 4102, the cartridge body 4108, and the valve through hole 4106. Without loss of generality, the dimensions of the valve may be tailored to the specific requirements of a given cartridge and instrument.

5 shows a partial view of a detection channel 5100 of a cartridge for sample testing, in accordance with some embodiments. The detection chamber panel includes a plurality of detection chambers, such as represented by 5102, which may be PCR amplification chambers, each fluidically coupled to a reagent chamber, such as PCR chamber 5104. A fluid conduit, such as indicated at 5106, provides dedicated fluidic communication between the detection chamber through a PCR valve, such as valve 5108, and a bubble trap, such as bubble trap 5110. The extraction chamber 5112 of the cartridge is shown in the figure for completeness and to provide a sense of perspective.

6A-6D show components of an optical coupler 6100 for use in an optical unit for an instrument, according to some embodiments. FIG. 6A shows several optical sources assembled to an excitation optics setup 6102 including filters, lenses, and optical fibers for delivering an excitation signal to the cartridge's detection chamber 6124 as described herein. The optical source may include a set of one or more (eg, four) light emitting diodes (LEDs), lasers, or any other type of monochromatic or quasi-monochromatic light sources 6106, 6108, 6110, and 6112. The LEDs can be configured to provide excitation signals for different fluorescent emitting tags. Each fluorescent emissive tag may represent a specific target nucleic acid portion to be detected. In the described excitation optics setup, the filter in front of each LED serves as a bandpass filter for the excitation light to prevent crosstalk and includes an antireflection (AR) coating to prevent fringing and other etalon effects in the optical module. You may. To combine and multiplex the different LEDs used, some embodiments may include beam splitter configurations that include dichroic or birefringent elements that transmit light at one wavelength or polarization and reflect light at a different wavelength or polarization. .

The excitation optics setup also includes a coupler 6114 to couple the light from the LEDs into a bundle of excitation fibers 6116. In some embodiments, without limitation, a bundle of excitation fibers 6116 may include 8 fibers (2 for each detection chamber). In some embodiments, the excitation optical setup may include fewer or more optical fibers. Moreover, in some embodiments, the excitation optics setup may be a free space excitation optics setup without optical fibers.

As shown in the figure, the two excitation fibers 6118 and 6120 may converge in each detection chamber 6124 of the detection chamber panel 6104. The two excitation fibers converge at an angle to each other, and the condensing lens 6122 collects the optical signal emitted from the detection chamber 6124. In some embodiments, a condensing lens 6122 is disposed between the two excitation fibers so that the condensing lens receives the cone of the signal emitted from the sample. In some embodiments, the condensing setting is such that the cone of emitted signal collected by the condensing lens has a reduced amount of excitation light scattered from the excitation fiber and a reduced amount of stray light from the background of the sample port.

A condensing lens receives the cone of light emitted from the sample and focuses it to detector 6126. The detector converts the optical signal into an electrical response signal that can be processed by the instrument's processor circuitry.

FIG. 6B shows the excitation optics setup 6102 of FIG. 6A , further comprising a detector having a pickup component 6200 for engaging the detection chamber panel of the cartridge and a connector 6202 for providing a response signal. The pick-up component 6200 may be configured to receive a bundle comprising excitation fibers 6116 that fit into the detection chamber panel of the cartridge and transmit an excitation signal. The pickup component 6200 also serves as a mount for the detector and connector 6202 and can be shaped and sized to reduce the amount of stray light reaching the detector.

6C shows a schematic diagram of an optical module 6300 coupled to a detection chamber panel 6316 of a cartridge, in accordance with some embodiments. The drawing shows the elements of an optical module (e.g., detector 6302, connector 6304, light source 6306, fiber bundle 6308) as described above assembled together in pickup component 6310. Also shown is a valve access port 6312 for an actuator of the instrument to access the valve of the fluid conduit into the detection chamber 6314.

6D shows a detector set 6400 having a filter assembly 6402 disposed adjacent to a silicon detector 6404, in accordance with some embodiments. In some embodiments, filter assembly 6402 includes an emission filter with a coating optimized to reduce internal reflection and other undesirable etalon effects. In some embodiments, filter assembly 6402 may include a channel isolation mask 6406 to prevent optical crosstalk between different filters in the assembly. For example, in some embodiments, a channel isolation mask 6406 is disposed within filter assembly 6402 to prevent crosstalk. It is uniform between filters.

7A-7AF show a sequence of steps in a sample testing method using a cartridge and instrument in accordance with some embodiments. Any one of the devices and components of the testing architecture disclosed herein may at least partially perform steps 7A-7AF. For example, at least some of Steps 7A-7AF, as disclosed herein (see FIG. 1 ), may include inserting a cartridge into an instrument and coupling an excitation signal from an optical source to the cartridge with an optical coupler. In one embodiment, the cartridge includes one or more structural features that facilitate insertion and/or alignment of the cartridge into and/or an instrument. Such features may include, for example, ridges, holes, dimples, divots, chamfers, and the like. In some embodiments, when the cartridge is inserted into the instrument with the sample port located below the centerline intersecting the cartridge, the orientation of the cartridge provides for sample movement from the sample port to the extraction chamber in the opposite direction of gravity.

The optical coupler may include a set of detectors that generate a response signal when a selected label in the sample receives an excitation signal. The response signal can be received and analyzed, and the results for the sample test are provided by the processor circuitry executing the instructions stored in the instrument's memory circuitry (see Figure 1). The cartridge may contain an extraction chamber, a detection chamber, and a sample suspected of containing the target nucleic acid. In some embodiments, at least a portion of the steps may be performed using a pump of the instrument's pneumatic system coupled with the cartridge through a gas supply port air manifold (see FIGS. 1 and 2 ). In some embodiments, a method consistent with this disclosure may include at least one of the steps of steps 7A through 7AF performed in any order. The series of steps shown in FIGS. 7A to 7AF include various combinations of open and close valves to at least partially perform steps 7A to 7AF, the open valve being designated as an open source, and the close valve being designated as a line. It is represented by a circle passing through it. Also in FIGS. 7A-7AF , liquid reagent positions and their movements, impacts or pushes on various canisters by various actuators, various shading patterns representing the positions of the various liquids as they move through features of the cartridge and steps 7A through 7A are illustrated in FIGS. In 7AF, it is indicated by the position of the various canisters when actuated by the various actuators. In addition, an embodiment according to the present invention may include one or more steps performed simultaneously, quasi-simultaneously, or temporally overlapping.

Step 7A includes placing the cartridge in the instrument.

Step 7B includes closing all valves of the cartridge except for each PCR valve located between each PCR reagent chamber and each detection chamber.

Step 7C occurs for a cartridge having a sample port that receives a liquid sample and includes opening certain valves associated with the sample port to prepare to fill the extraction chamber.

Step 7D occurs for a cartridge having a sample port to receive a liquid sample, actuating a force against the reagent canister 1, moving fluid from the reagent canister 1 to the extraction chamber, and extracting from the sample port. moving the sample into the chamber, and moving the PMP into the extraction chamber. It will be appreciated that the PMP may be stored in the chamber or canister, or in some embodiments already located in the extraction chamber.

Step 7E occurs for a cartridge having a port receiving a swab with a sample and includes opening a valve to allow fluid to flow from the reagent canister to contact the swab.

Step 7F occurs for a cartridge having a port that receives a swab with sample, pushing the canister 1 to release the fluid into the port where the swab is received (swap not shown in FIG. 7F), canister 1 ) to the swap port.

Step 7G includes moving fluid in contact with the swab to the extraction chamber, in this embodiment, the fluid moves through the chamber with the dried PMP and carries the PMP along with the fluid to the extraction chamber. Thus, the sample and PMP are now in the extraction chamber.

Step 7H includes closing all valves except for the valve between the PCR reagent chamber and the detection chamber and mixing the components of the extraction chamber (e.g., by activating a sonicator in the instrument so that the sonicator is extracted). near the chamber or in contact with the surface of the cartridge in the extraction chamber).

Step 7I involves collecting the PMP (eg, using a local magnetic field), generally by collecting the PMP against the walls of the extraction chamber.

Step 7J includes draining or removing fluid from the extraction chamber to the waste chamber through a waste path.

Step 7K includes pushing the canister 2 so that the contents can be ejected, and moving the contents of the canister 2 into the extraction chamber. The reagent canister 2 has a wash solution, for example a wash buffer.

Step 7L includes closing all valves other than those between the PCR reagent chamber and the detection chamber and mixing the components of the extraction chamber (eg, by activating a sonicator).

Step 7M involves collecting (eg, by using a local magnetic field) magnetic beads (referred to as PMPs in the figures, but not intended to be limiting) in the extraction chamber.

Step 7N includes removing fluid from the extraction chamber to the waste chamber through the waste path.

Step 70 includes pushing the canister 3 so that its contents can be ejected, and moving the contents of the canister 3 into the extraction chamber. The canister 3 may contain, for example, a cleaning solution.

Step 7P includes closing all valves other than those between the PCR reagent chamber and the detection chamber, and mixing the contents of the extraction chamber (eg, using a sonicator).

Step 7Q generally involves collecting the PMP by collecting the PMP against the walls of the extraction chamber (eg, by using a local magnetic field).

Step 7R includes removing fluid from the extraction chamber to the waste chamber via the waste path.

Step 7S includes closing all valves other than the valve between the PCR reagent chamber and the detection chamber.

Step 7T involves opening the valve to open the reagent canister 4 pathway, turning the heater 'on' and forcing heated air through the extraction chamber. In some embodiments, step 7T includes applying a local magnetic field to maintain the PMP in the extraction chamber.

Step 7U includes closing all valves other than those between the PCR reagent chamber and the detection chamber.

Step 7V includes pushing the canister 4 so that its contents can be ejected, and moving the contents of the canister 4 into the extraction chamber. Canister 4 may contain an elution medium.

Step 7W includes obtaining, using the metering function, a defined volume of fluid in the brewing chamber, wherein at least a certain defined amount of fluid exits the brewing chamber at an outlet port approximately at the centerline of the brewing chamber, and excess fluid is returned to the canister. (4) or moved to the overflow chamber.

Step 7X includes closing all valves other than those between the PCR reagent chamber and the detection chamber, and mixing the contents of the extraction chamber (eg, using a sonicator).

Step 7Y involves collecting the PMP (eg, using a local magnetic field). The PMP remains collected in the extraction chamber for the remainder of the process.

Step 7Z involves passing fluid (typically a defined amount of elution buffer in which nucleic acids are eluted from the PMP surface) into the PCR reagent chamber and the connecting fluid conduit.

Step 7AA includes opening the indicated valve in the connecting fluid conduit to the waste chamber to meter and define the volume of fluid in the PCR reagent chamber.

Step 7AB includes closing all valves, including the valve between the PCR reagent chamber and the detection chamber, and sonicating the PCR reagent chamber.

Step 7AC includes opening a valve between the PCR reagent chamber and the detection chamber.

Step 7AD includes transferring fluid from the PCR reagent chamber to the detection chamber.

Step 7AE includes closing all valves between the PCR reagent chamber and the detection chamber, and performing a cycle process in each detection chamber to amplify the nucleic acid. Any PCR process can be performed in a PCR/detection chamber, including but not limited to thermal cycling, isothermal reactions, PT-PCR, rapid multiplex PT-PCR, Taq-man® multiplex real-time PT-PCR reactions, and the like. It should be understood that it can. In some embodiments, a PCR process is performed in a PCR/detection chamber assay for the qualitative detection and discrimination of target nucleotides, such as nucleotides from a pathogen of interest. In some embodiments, the PCR process performed in the PCR/detection chamber is performed under optimized conditions to generate amplicons for the nucleotide of interest, such as the target viral nucleotide (if present) and any process controls present in the sample.

In some embodiments, step 7AE may also include detecting (eg, detecting, identifying, and/or distinguishing) the presence or absence of an amplicon of the nucleic acid of interest. Such detection may be performed concurrently with or after the PCR amplification process. In some embodiments, detecting the amplification product in the detection chamber is accomplished by illuminating the detection chamber with an excitation signal and detecting the presence or absence of any signal from an amplification target of interest with an excitation signal from an optical source via an optical coupler. and probing the detection chamber.

Step 7AF includes releasing all valves and actuators to allow removal of the cartridge from the instrument.

8 is a flow diagram illustrating steps of a sample testing method 800 in accordance with some embodiments. Method 800 may be performed at least in part by any one of the devices and components of a testing architecture as disclosed herein. For example, at least some of the steps of method 800 may include inserting a cartridge into an instrument as disclosed herein (see FIG. 1 ) and coupling an excitation signal from an optical source to the cartridge with an optical coupler. there is. The optical coupler may include a set of detectors that generate a response signal when a selected label in the sample receives an excitation signal. Response signals may be received, analyzed, and results for sample tests provided by processor circuitry executing instructions stored in the instrument's memory circuitry (see Figure 1). The cartridge may contain an extraction chamber, a detection chamber, and a sample suspected of containing the target nucleic acid. In some embodiments, at least some of the steps of method 800 may be performed using a pump of a pneumatic system of an instrument coupled to a cartridge via a gas supply port air manifold (see FIGS. 1 and 2 ). In some embodiments, a method included in this disclosure may include at least one of the steps of method 800 performed in any order. Moreover, embodiments consistent with this disclosure may include one or more of the steps of method 800 being performed concurrently, quasi-simultaneously, or overlapping in time.

Step 802 includes providing an extraction chamber, a detection chamber, and a cartridge containing a sample suspected of containing the target nucleic acid. In some embodiments, step 802 includes inserting the swab into a sample port on the cartridge. In some embodiments, step 802 includes rinsing the swab with lysis buffer after insertion into the sample port and prior to transferring the sample to the extraction chamber.

Step 804 includes moving a plurality of magnetic particles and a fluid through a first port of the cartridge into an extraction chamber. In some embodiments, the sample moves from the sample port into the extraction chamber in the opposite direction of gravity. In some embodiments, the fluid comprises a lysis buffer configured to release nucleic acid material from cellular or pathogenic components of the sample. Pathogen components can be bacteria, viruses, prions, spores, and the like.

Step 806 includes using a magnetic field to capture a plurality of magnetic particles complexed with nucleic acids when the extraction chamber is essentially or substantially fluid free. In some embodiments, step 806 also includes introducing a gas into the extraction chamber at a temperature of about 35° C. or greater.

Step 808 includes introducing a known volume of elution medium into the extraction chamber through the second port of the cartridge. In some embodiments, the second port of the cartridge is identical to the first port. In some embodiments, the second port of the cartridge is different than the first port.

In some embodiments, step 808 includes heating a region upstream of the extraction chamber and passing gas through the region to heat the gas and introducing the heated gas into the extraction chamber. In some embodiments, step 808 includes heating the gas into the extraction chamber to a temperature of greater than or equal to about 35° C. and less than or equal to 90° C.

Step 810 includes releasing a plurality of magnetic particles complexed with nucleic acids from the magnetic field into an elution medium to release nucleic acids from the plurality of magnetic particles. In some embodiments, step 810 includes moving the lysis buffer and sample out of the extraction chamber. In some embodiments, step 810 includes moving the cleaning solution to the extraction chamber. In some embodiments, step 810 includes releasing the magnetic particles complexed with the nucleic acids from the magnetic field into the wash solution. In some embodiments, step 810 includes sonicating the extraction chamber to release the nucleic acids complexed with the magnetic particles into the elution medium.

Step 812 includes trapping the plurality of magnetic particles with a magnetic field to maintain the plurality of magnetic particles in the extraction chamber. In some embodiments, step 812 includes moving the cleaning solution out of the extraction chamber. In some embodiments, method 800 includes repeating steps 810 and 812 with a second wash solution.

Step 814 includes moving elution medium and nucleic acid from the extraction chamber to a downstream chamber for contact with reagents for nucleic acid amplification. In some embodiments, step 814 is performed through a third port of the cartridge, which is different from the first port of the cartridge and the second port of the cartridge. In some embodiments, step 814 includes mixing the nucleic acid and an elution medium with a reagent for amplification and detection (eg, detection, identification, and/or discrimination) of amplicons of the nucleic acid. In some embodiments, the downstream chamber is a master mix chamber upstream of the detection chamber and step 814 includes sonicating the master mix chamber to form a process fluid. In some embodiments, step 814 includes heating the detection chamber.

In some embodiments, the detection chamber includes four separate detection chambers, and step 814 includes introducing an amount of elution medium and nucleic acid from the extraction chamber into each separate detection chamber.

Step 816 includes amplifying the nucleic acid and detecting the amplification product in a detection chamber. In some embodiments, step 816 includes irradiating the detection chamber with an excitation signal from an optical source through an optical coupler. In some embodiments, step 816 includes illuminating the detection chamber with an excitation signal.

As used herein, the phrase "at least one of" preceding a series of items, with the terms "and" or "or" to separate any item, Modify the list as a whole, not each component of (eg each item). The phrase “at least one of” does not require selection of at least one item; Rather, the phrase permits a meaning that includes any one of the items, and/or at least one of any combination of items, and/or at least one of each item. By way of example, "at least one of A, B, and C" or "at least one of A, B, or C" )" is only A, only B, or only C, respectively; any combination of A, B, and C; and/or at least one of each of A, B, and C.

To the extent "include", "have" or similar terms are used in the description or claims, such terms shall be construed as "comprises" when the term "comprise" is used as a transitional word in a claim. It is intended to be inclusive in a manner similar to "comprising". The word "exemplary" is used herein "serving as an example, instance, or illustration." Any embodiment described herein as “exemplary” should not necessarily be construed as preferred or advantageous over other embodiments.

Reference to a singular element is not intended to mean “one and only one” unless specifically stated, but rather “one or more”. All structural and functional equivalents to the various structural elements described throughout this disclosure, which are known or become known to those skilled in the art, are expressly incorporated herein by reference and are intended to be incorporated into the subject technology. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is expressly recited in the above description.

Although this specification contains many details, they should not be construed as limitations on the scope of what may be claimed, but rather as a description of specific implementations of the subject matter. Certain features that are described in this specification in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, even if features are described above as operating in particular combinations, and even if initially so claimed, one or more features from a claimed combination may in some cases be excluded from the combination, a claimed combination being a subcombination or a variant of a subcombination. may be about

Although subject matter in this specification has been described in terms of certain aspects, other aspects may be implemented and are within the scope of the following claims. For example, while actions are shown in the figures in a particular order, it should be understood that such acts are performed in the particular order shown or in a sequential order, or require that all acts illustrated be performed in order to achieve a desired result. should not be The actions recited in the claims can be performed in a different order and still achieve the desired results. In one example, the processes depicted in the accompanying figures do not necessarily require the specific order or sequential order presented to achieve desired results. Multitasking and parallel processing can be advantageous in certain situations. Moreover, the separation of various system components in the aspects described above should not be understood as requiring such separation in all aspects, and the described program components and systems may generally be integrated together into a single software product or packaged into multiple software products. It should be understood that there is Other variations are within the scope of the following claims.

In one aspect, a method can be an action, command, or function, and vice versa. In one aspect, a claim includes one or more other claims, one or more words, one or more sentences, one or more phrases, one or more paragraphs, and/or words recited in one or more claims (e.g., commands, actions, functions, or elements). ) may be modified to include some or all of

To illustrate the interchangeability of hardware and software, items such as various illustrative blocks, modules, components, methods, operations, instructions, and algorithms have been described generally in terms of functionality. Whether these functions are implemented in hardware, software, or a combination of hardware and software depends on the specific application and design constraints that apply to the overall system. Skilled artisans may implement the described functionality in varying ways for each particular application.

Aspect, said aspect, other aspect, some aspect, one or more aspects, implementations, said implementations, other implementations, some implementations, one or more implementations, embodiments, said embodiments, other embodiments, some embodiments, one or more embodiments, References to a configuration, the above configuration, other configurations, some configurations, one or more configurations, subject technology, disclosure, this disclosure, other variations thereof and similar phrases are for convenience only, and the disclosure with respect to such phrase(s) is essential to the subject technology or It is not meant that such disclosure applies to all configurations of the subject technology. A disclosure relating to such phrase(s) may apply to all or one or more configurations. A disclosure relating to such phrase(s) may provide one or more examples. A phrase such as an aspect or some aspect may refer to one or more aspects and vice versa, which similarly applies to other phrases described above.

Masculine pronouns (eg, he) include the feminine and neuter genders (eg, she or it) and vice versa. “Some” means one or more. Topic and subheadings in underlined and/or italic type are used for convenience only and do not limit the subject technology and are not to be referred to in connection with the interpretation of the subject technology description. Relative terms, such as first and second, may be used to distinguish one entity or action from another without necessarily requiring or implying an actual such relationship or order between such entities or actions. All structural and functional equivalents to the various structural elements described throughout this disclosure that are known or become known to those skilled in the art are expressly incorporated herein by reference and are intended to be incorporated into the subject technology. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is expressly recited in the above description. Unless an element is explicitly recited using the phrase “means for” or, in the case of a method claim, an element is not recited using the phrase “step for”. provided that any element of the claim is not subject to 35 U.S.C. §112, sixth paragraph.

Claims (22)

As a system,
cartridge; and a mechanism configured to receive the cartridge;
The cartridge is:
(i) a plurality of chambers, the plurality of chambers including an extraction chamber and a detection chamber;
(ii) a plurality of reagent canisters, each reagent canister of the plurality containing a reagent; and
(iii) a plurality of magnetic particles once introduced into the extraction chamber and retained therein;
The mechanism is:
(i) a first sonicator movable in at least one of xyz coordinates;
(ii) a magnetic field positioned to capture the plurality of magnetic particles in the extraction chamber; and
(iii) an optical unit for illumination of the detection chamber and detection of signals therefrom. The method of claim 1,
A portion of the plurality of reagent canisters is in fluid communication with the extraction chamber. According to claim 1 or claim 2,
wherein the reagent in a portion of the plurality of reagent canisters is a liquid reagent. The method according to any one of claims 1 to 3,
All or some of the plurality of reagent canisters are in fluid communication with a dedicated gas source. The method according to any one of claims 1 to 4,
The system of claim 1 , wherein at least one reagent canister of the plurality of reagent canisters includes a frangible membrane and a piercing element. The method of claim 5,
The system of claim 1 , wherein the piercing element comprises a hollow conduit. The method of claim 6,
Gas from a dedicated gas source is moveable from the gas source to a reagent canister through the hollow conduit. According to any one of claims 1 to 7,
wherein each reagent canister interacts with an actuator of the instrument. The method of claim 8,
A system in which each reagent canister interacts with a dedicated actuator. As a cartridge:
(i) a plurality of chambers, the plurality of chambers including an extraction chamber and at least one detection chamber;
(ii) an inlet port for engaging a gas supply source;
(iii) a plurality of reagent canisters, each reagent canister of the plurality containing a reagent, each reagent canister including a frangible stopper and a piercing element having an opening in fluid communication therewith; and
(iv) a cartridge comprising a plurality of magnetic particles in a first position and movable to said extraction chamber. The method of claim 10,
the plurality of chambers further comprising one or more sample ports in fluid communication with the extraction chambers. The method of claim 11,
The cartridge of claim 1 , wherein the one or more sample ports are configured to receive a swab containing a sample. The method of claim 10,
The cartridge of claim 1 , wherein the one or more sample ports include a liquid port configured to receive a liquid sample. The method of claim 13,
wherein the liquid port is configured to identify the presence of a liquid sample by obstruction of total internal reflection of a light beam when a liquid sample is present in the liquid port. According to any one of claims 10 to 14,
wherein the plurality of reagent canisters include reagent canisters having a dissolution medium in fluid communication with one or more sample ports. The method according to any one of claims 10 to 15,
wherein the plurality of reagent canisters include reagent canisters having a volume of elution medium in fluid communication with the extraction chamber. The method according to any one of claims 10 to 16,
The cartridge of claim 1 , wherein the at least one detection chamber includes two, three, or four detection chambers. According to any one of claims 10 to 17,
wherein said at least one detection chamber is in fluid communication with a reagent chamber containing a reagent for amplifying a nucleic acid. According to claim 17 or claim 18,
The at least one detection chamber is in fluid communication with dedicated bulb chambers containing lyophilates of reagents for amplification, and each lyophilate in each dedicated bulb chamber is a different cartridge. As a cartridge,
(i) an extraction chamber configured to receive and/or dispense a precise amount of liquid;
(ii) a plurality of reagent canisters, each reagent canister containing a reagent; and
(iii) a plurality of magnetic particles in a first position and movable into the extraction chamber, once moved into the extraction chamber, the plurality of magnetic particles are retained therein. A method for identifying the presence or absence of a target nucleic acid in a sample, comprising:
i. providing a cartridge containing an extraction chamber, a detection chamber disposed downstream from the extraction chamber, and a sample suspected of containing a target nucleic acid;
ii. moving a sample, a plurality of magnetic particles, and a fluid into the extraction chamber;
iii. entrapping the plurality of magnetic particles complexed with the target nucleic acid, if present, with a magnetic field in the essentially fluid-free extraction chamber and introducing a gas at a temperature of about 35° C. or greater into the extraction chamber;
iv. introducing a known volume of elution medium into the extraction chamber;
v. releasing the plurality of magnetic particles complexed with the target nucleic acid, when present, from the magnetic field to the elution medium to release the target nucleic acid from the plurality of magnetic particles;
vi. trapping the plurality of magnetic particles with a magnetic field to retain the plurality of magnetic particles in the extraction chamber;
vii. moving the elution medium and the target nucleic acid from the extraction chamber to a downstream chamber for contact with a reagent for amplifying the target nucleic acid; and
viii. A method comprising amplifying the target nucleic acid and detecting an amplification product in the detection chamber. 21. A kit comprising the cartridge of any one of claims 10-20, a pipette and user instructions, wherein the pipette comprises an overflow chamber and is configured to dispense a fixed sample volume.

Images